Overall, the prevalence of α-thalassemia is 22.6%. The highest α-thalassemia prevalence was observed in Vietnam (51.5%) followed by Cambodia (39.5%), Laos (26.8%), Thailand (20.1%), and Malaysia (17.3%). No publication bias was detected. Conclusions: This meta-analysis suggested that a high prevalence of α-thalassemia occurred in selected Southeast Asia countries. This meta-analysis data are useful for designing thalassemia screening programs and improve the disease management.Alpha(α)-thalassemia is a blood disorder caused by many types of inheritable α-globin gene mutations which causes no-to-severe clinical symptoms, such as Hb Bart’s hydrops fetalis that leads to early foetal death. Therefore, the aim of this meta-analysis was to provide an update from year 2010 to 2020 on the prevalence of α-thalassemia in Southeast Asia. A systematic literature search was performed using PubMed and SCOPUS databases for related studies published from 2010 to 2020, based on specified inclusion and exclusion criteria. Heterogeneity of included studies was examined with the I2 index and Q-test. Funnel plots and Egger’s tests were performed in order to determine publication bias in this meta-analysis. Twenty-nine studies with 83,674 subjects were included and pooled prevalence rates in this meta-analysis were calculated using random effect models based on high observed heterogeneity (I2 > 99.5,  This results in the elongation of the α-globin chain protein and causes severe anemia with serious complications that include liver impairment, cardiac disease and endocrine disorder . Pu. Pu19]. All search results were screened by two investigators, and all potential studies were independently reviewed to be included in this meta-analysis. The main inclusion criteria were: (1) studies published in English in which the prevalence of α-thalassemia  in Southeast Asian countries were reported; and (2) the study was a peer-reviewed publication. Studies that did not report cross-sectional, observational, cohort, or prevalence of α-thalassemia were excluded. We identified additional eligible studies based on references that were cited in the relevant articles. When publications overlapped, only the study with the largest or the most recent data was included in this meta-analysis. Data, including first author’s name, publication year, country, sample size, and prevalence of α-thalassemia of the included studies, were extracted and documented by the reviewing investigators. A total number of 278 articles were included in this meta-analysis . The stu2 index (represented as percentage) and Q-test  that indicated heterogeneity between studies. It was reported that an I2 value > 75% and Q-test with a p-value < 0.1 was regarded as high heterogeneity [The prevalence of α-thalassemia was calculated for each study with the number of reported α-thalassemia cases as the numerator and the total sample size as the denominator. Homogeneity across studies was investigated with the Iogeneity ,21. A raogeneity ,23. All ogeneity .Twenty-nine studies with a total number of 83,674 subjects were included in this meta-analysis after a detailed assessment of records obtained from the database and additional searching. These studies were published between January 2010 and October 2019. Among all included studies, two were carried out in Cambodia, three in Laos, five in Malaysia, 20 in Thailand, and two in Vietnam . The mai2 > 99.5% and p-value < 0.001 in overall and all subgroups except Cambodia   . In the e < 0.1) .t-value = 1.24 with a p-value = 0.112.Funnel plots and Egger’s tests were performed to estimate the publication bias of the included literature. The shape of the funnel plot revealed obvious evidence of symmetry . The valThis study is the first to report the prevalence of α-thalassemia in the Southeast Asia region over the past 10 years (2010–2020). We did not obtain any α-thalassemia-related studies that fulfilled our inclusion and exclusion criteria from other Southeast Asian countries, including Brunei, Indonesia, Myanmar, Philippines, Singapore, and Timor-Leste. Using a random effects model, the overall prevalence rate of α-thalassemia in the included countries was 22.6%, which indicated a significant reduction of ~50% of the prevalence in the Southeast Asia region since 2008. The World Health Organization had reported the α-thalassemia prevalence as 44.6% in 2008 . India aThe high prevalence of α-thalassemia in the past was due to the lack of knowledge regarding the seriousness of this disease among the populations in these countries, especially those living in rural areas with limited access to education and those who could not afford to obtain the proper education similar to that found in urban areas. α-thalassemia is an inherited disease and the mutations may pass from parent to child, affecting the haemoglobin production. Hence, the educational and screening campaigns regarding this disease conducted by the representative bodies (either government or non-government organization) have successfully reduced the prevalence of α-thalassemia in the Southeast Asia region.t-value = 1.24; p-value = 0.112) in which the publication bias did not significantly exist in this meta-analysis. Therefore, we concluded that there was no publication bias detected in this meta-analysis.Random effects models were used, which are based on the assumption that the true effect could vary between studies . The exiWhen stratified according to country, Vietnam has the leading α-thalassemia prevalence rate of 51.5%. The high prevalence rate is probably due to one of the observational studies conducted in Vietnam focusing on the country’s minority ethnic groups and thus, this likely skewed the actual prevalence rate ,5. TherePlasmodium [Thalassemia patients suffers from anemia caused by the imbalance of globin chains and impairment of haemoglobin solubility of erythrocytes. The reduced globin chains were shown to impair the cytoadherence of asmodium ,55. Thesasmodium ,56. Hencasmodium .There are several limitations that should be addressed in this meta-analysis. Firstly, only data from certain Southeast Asian countries were available to be included in this meta-analysis; therefore, the calculated α-thalassemia-related prevalence rate in this study was specific to selected Southeast Asian regions. Besides, only studies from 2010 to 2020 were included in this meta-analysis, and it is possible for studies published before the year 2010 that meet the inclusion criteria but were not included in this meta-analysis, as this study focused on the prevalence rates from recent past 10 years only. We also did not include ethnic stratification because the majority of the studies included in this analysis did not report the ethnic group in the subject population. Alpha thalassemia genotype stratification was not be performed due to inconsistencies in the reporting of genotypes in the studies included in this analysis.This is the first meta-analysis that investigated α-thalassemia prevalence in Southeast Asian countries, and the findings suggest high prevalence of α-thalassemia events in certain countries which warrants attention as α-thalassemia major could cause severe health complications and impose a substantial burden to the health authority and families. The data in this meta-analysis may be beneficial to the representative bodies in designing educational and screening campaigns regarding this disease in order to further reduce α-thalassemia rates in these countries.


Mucopolysaccharidosis type IIIA  is one of the most severe lysosomal storage disorder (LSD) caused by the inherited deficiency of sulfamidase, a lysosomal sulfatase enzyme involved in the stepwise degradation of heparan sulfates (HS). MPS-IIIA patients show multisystemic problems, including a strong impairment of central nervous system (CNS), mild somatic involvement, and ocular manifestations that result in significant visual impairment. Despite the CNS and somatic pathology have been well characterized, studies on visual system and function remain partially explored. Here, we characterized the retina morphology and functionality in MPS-IIIA mouse model and analyzed how the SGSH deficiency affects the autophagic flux. MPS-IIIA mice exhibited a progressive retinal dystrophy characterized by significant alterations in visual function. The photoreceptor degeneration was associated with HS accumulation and a block of autophagy pathway. These events caused a reactive microgliosis, and a development of apoptotic processes in MPS-IIIA mouse retina. Overall, this study provides the first phenotypic spectrum of retinal disorders in MPS-IIIA and significantly contributes for diagnosis, counseling, and potential therapies development. Mucopolysaccharidoses (MPSs) belong to a group of lysosomal storage disorders (LSDs) caused by the deficiency of lysosomal enzymes involved in the catabolism of complex polysaccharides, the glycosaminoglycans (GAGs). GAGs derived from the degradation products of proteoglycans and are localized on cell surfaces and in the intracellular matrices. MPSs are classified in seven families  basing on wide diversity of phenotype, some are lethal within the first period of life and others have no significant impact on life duration. MPSs patients show chronic and progressive clinical symptoms with multisystem involvement including visceral, skeletal, muscular, neurological, and ocular manifestations. Ophthalmic problems are very common in MPS patients with variable severity and age of onset. They are associated with the accumulation of GAGs and comprise corneal clouding, glaucoma, atrophy of optic nerve, and retinal dysfunction. MPS-III disorders  represent the most prevalent form of MPS due to the deficiency of four different lysosomal enzymes involved in the degradation of heparan sulfates (HSs). Patients of different MPS-III subtypes show a very severe neuropathology including behavioral problems  and only mild somatic manifestations. Most of MPS-III patients show a severe retinopathy associated with pigmentary retinal degeneration associated to changes in electroretinogram, but few documented cases present with corneal opacity and optic nerve atrophy . Among S2 evoke responses originated in the mesopic range , therefore these data support the possibility that the recorded decrease of ERG was due to defects in both rod- and cone-derived components. We then examined whether the retinal functional degeneration in 3 months old MPS-IIIA mice is associated with morphological alteration of photoreceptor cells. This analysis was performed by measuring the density of both cones and rods in retinal sections labeled with anti-c-Arrestin and anti-Rhodopsin antibodies, which mark respectively cones and rods. Interestingly, we did not detect any significant alteration in rods and outer nuclear layer (ONL) thickness, but we observed a significant decrease in cone densities in MPS-IIIA mice compared to age-matched WT mice  mice at different time points: 3-, 6-, 9 months of age according to progression of neuropathology. Retinal function was first evaluated by standard electroretinographic (ERG) recordings in mice at 3 months of age. Our results showed a decrease of a-wave amplitude with a relatively preserved b-wave amplitude in MPS-IIIA mice compared to age-matched WT mice . Importa WT mice . OverallUsually, the earliest clinical symptom of retinal degeneration is an initial dysfunctional photoreceptor system. Subsequent degeneration of rods and cones leads to a complete loss of the visual field. To further explore the phenotypic consequences in the retina of MPS-IIIA, we expanded our morphological and functional analyses at later ages, focusing on 6- and 9 months old animals, in which pathological signs of neurodegeneration in the CNS are evident . NotablyTo gain insight into the retinal phenotype of MPS-IIIA mice, we aimed at determining photoreceptor cell death in retinal dystrophy of MPS-IIIA mice. To that purpose, we carried out cell death assessment by TUNEL assay that revealed a significant increase in the number of apoptotic cells in the retinal ONL of MPS-IIIA mice compared to age-matched WT mice at 3-, 6-, and 9 months of age , furtherIn order to assess if the photoreceptor degeneration is associated with HS storage in the same retinal compartment, we performed a quantitative measurement of GAGs in RPE of MPS-IIIA mice at different time points of retinal pathology. This analysis revealed a progressive increase of GAGs in RPE of MPS-IIIA mice from 3 to 9 months of age respect to the age-matched WT mice. In particular, at 9 months of age, we observed an increase of GAGs about twofold higher in the RPE from MPS-IIIA mice compared to age-matched WT mice . Since aThe relevance of the pathological consequences associated to HS accumulation in the CNS of MPS-IIIA mouse model has been recently gathered. Nevertheless, there is only little information available on the effects of HS accumulation on retina maintenance and function. Here, we show the first evidence of a specific relationship between HS accumulation and photoreceptor function and survival in mammals. We describe a detailed physiological and morphological characterization of visual impairment in MPS-IIIA mouse model trying to outline the causes of this dysfunction. In detail, we observed a progressive decrease of retinal functionality in MPS-IIIA mice by ERG experiments at different ages 3-, 6-, and 9 months. The reduction of a-wave at early time point (3 months of age) suggests that the retinopathy is primary caused by photoreceptors degeneration. This functional deterioration of photoreceptors is associated with a progressive, significative, and specific reduction of ONL thickness of MPS-IIIA mice retina that culminates at 9 months of age in which the animals also show a severe CNS degeneration. The latter conclusion was supported by a number of evidences, a special emphasis should be placed on the lack of a TUNEL-positive cells in the inner nuclear layer (INL), even in old mice, and the unaltered interneurons morphology as assessed by immunofluorescence. Similar conditions have been described in another mouse model of Sanfilippo syndrome like MPS-IIIB and clearly observed in MPS-IIIA patients but never analyzed in MPS-IIIA mouse model . Since tOverall these data not only confirmed the block of autophagy pathway and protein degradation impairment in the retina as observed in the CNS of MPS-IIIA mouse model, but our findings contribute in uncovering of the role of HS accumulation in photoreceptor survival and function. Finally, our results describe in the deep structural, functional, and biochemical aspects of retinal dysfunction as first pathological sign of CNS impairment in the MPS-IIIA mice highlighting the importance to carry out retinal assessment for a development of therapeutic approach for the treatment of neuropathology in MPS-IIIA disorder.–/–sgsh) employed in this study was maintained and analyzed as described previously . The following primary antibodies were used: mouse anti-Rhodopsin , rabbit anti-c-Arrestin , rabbit anti-Pax6 , rabbit anti-PCKα , mouse anti-GS , mouse anti-β-III tubulin , rabbit anti-Iba-1 , rat anti-Lamp1 . All incubations were performed overnight at 4°C. After washing with 1% PBS, slides were incubated with the following secondary antibodies: Alexa 594 goat anti-rat/rabbit/mouse  or Alexa 488 goat anti-rabbit/mouse  and DAPI  for 1 h at room temperature, then the slides were washed with 1% PBS and mounted with PBS/glycerol or dehydrated before mounting . Sections were observed with a Leica DM-6000 microscope and then confocal images were acquired using the LSM710 Zeiss Confocal Microscopy system. The number of marker-positive cells was generally evaluated in the central area of the retina by manual counts with a Leica DM-6000 microscope, with the objective Leica ∞/0.17/D, HCX PL FLUOTAR, 40X/0.75 that has an area of 0.31 mmMouse eyes were enucleated and the RPE was separated from the retina. RPE samples were lyzed by using RIPA buffer  with inhibitors cocktail . The concentration of total protein was determined by Bradford analysis and quantified by using a NanoDrop ND-8000 spectrophotometer (NanoDrop Technologies). The extracted proteins (20 μg) were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a PVDF membranes . After blocking with Tween 0.1%-Tris-buffered saline containing 5% non-fat milk or 5% bovine serum albumin  for 1 h at room temperature, membrane filters were incubated overnight at 4°C with primary antibodies. For Western blot analysis, the following antibodies were used: anti-Lamp1 , anti-Nbr1 , anti-LC3 , and anti-Gapdh . After washing with Tween 0.1%-Tris-buffered saline, the membranes were incubated for 1 h at room temperature with the following secondary antibodies: goat anti-rabbit IgG antibody, HPR conjugate, and goat anti-mouse IgG antibody HPR conjugate . Western blot bands were revealed using a chemiluminescence digital imaging system  and quantified using ImageJ software.–4 to 20 cd s/m2 generated. ERGs and b-wave thresholds were assessed using the following protocol: eyes were stimulated with light flashes increasing from −5.2 to + 1.3 log cd s/m2 (which corresponds to 1 × 10–5.2 to 20.0 cd s/m2) in scotopic conditions. The log unit interval between stimuli was 0.3 log from −5.4 to 0.0 log cd s/m2, and 0.6 log from 0.0 to + 1.3 log cd s/m2. For ERG analysis in scotopic conditions, the responses evoked by 11 stimuli (from −4 to + 1.3 log cd s/m2) with an interval of 0.6 log unit were considered. To minimize the noise, three ERG responses were averaged at each 0.6 log unit stimulus from −4 to 0.0 log cd s/m2 while one ERG response was considered for higher (0.0- + 1.3 log cd s/m2) stimuli. The time interval between stimuli was 10 s from −5.4 to 0.7 log cd s/m2, 30 s from 0.7 to + 1 log cd s/m2, or 120 s from + 1 to + 1.3 log cd s/m2. a- and b-wave amplitudes recorded in scotopic conditions were plotted as a function of increasing light intensity (from −4 to + 1.3 log cd s/m2). The photopic ERG was recorded after the scotopic session by stimulating the eye with 10 flashes of 10 ms with 20.0 cd s/m2 light intensity on a constant background illumination of 50 cd/m2.Scotopic and photopic electrophysiological recordings were performed as described . A NatioIn Situ Cell Death Detection Kit, Fluorescein (Roche 11684795910) following the manufacturer’s directions. Twelve-micrometer cryosections were collected on slides and submit to TUNEL assay. To consider the presence of unspecific results, some retina sections were incubated with the reaction mix without TUNEL reaction enzyme. Sections were observed with a Leica DM-6000 microscope and then confocal images were acquired using the LSM710 Zeiss Confocal Microscopy system. The number of TUNEL-positive cells was evaluated in the central part of the retina by manual counts with a Leica DM-6000 microscope, with the objective Leica ∞/0.17/D, HCX PL FLUOTAR, 40X/0.75 that has an area of 0.31 mm2.The number of apoptotic cells was analyzed by TdT-mediated dUTP nick end labeling (TUNEL), using the 2.The number of cones/area was manually estimated in at least 12 retina sections immunostained with c-Arrestin from each eye sample by using a Leica DM-6000 microscope, with the objective Leica ∞/0.17/D, HCX PL FLUOTAR, 40X/0.75 that has an area of 0.31 mm2), using Z-stacks in the wavelength of DAPI. The ONL thickness and OS length were manually measured using ImageJ software.At least 12 retina sections were imaged by confocal microscopy using a Leica TCS SPE 40x objective . The RPE/choroid were peeled from the eyecup and transferred to a tube; 5–50 mg of RPE was dissolved in 300 μL of sterile water and treated with proteinase k 50 μg/mL at 55°C for 2 h at 750 r/min. After centrifugation at 10,000 r/min for 10 min, DNA was quantified by using Nanodrop. GAGs were characterized using Blyscan Sulfated Glycosaminoglycans Assay (Biocolor B1000) manufacturer’s protocol.The datasets generated for this study are available on request to the corresponding authors.All studies on animals were conducted in strict accordance with the institutional guidelines for animal research and approved by the Italian Ministry of Health; Department of Public Health, Animal Health, Nutrition and Food Safety in accordance to the law on animal experimentation (DL 27/01/1992 No. 116). Furthermore, all animal treatments were reviewed and approved in advance by the Ethics Committee of TIGEM Institute .in vivo data. DI, GG, EM, VM, FS, and EN performed in vivo experiments. DI and GG performed the molecular biology studies and analyses on imaging data. IC and NS supervised the work and wrote the manuscript. The authors declare not to have any financial competing interests.DI, IC, and NS conceived the study, designed the experiments, and interpreted the data. AF contributed to experimental implementation and interpretation of the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P < 0.001) and was closely related to high TNM stage (P = 0.006) and histological grade (P = 0.004) and poor prognosis of patients (P < 0.001). In vivo and in vitro experiments confirmed that LINC02747 could promote the proliferation of ccRCC cells. We also found that LINC02747 regulated the proliferation of RCC cells by adsorbing miR-608. Subsequent mechanistic research showed that miR-608 is downregulated in ccRCC (P < 0.001), and overexpression of miR-608 inbibited the proliferation of RCC cells. Moreover, we found that TFE3 is a direct target gene of miR-608. MiR-608 regulated the proliferation of RCC cells by inhibiting TFE3. In conclusion, LINC02747 upregulates the expression of TFE3 by adsorbing miR-608, ultimately promoting the proliferation of ccRCC cells. The above findings indicate that LINC02747 acts as an oncogene in ccRCC and may be developed as a molecular marker for the diagnosis and prognosis of ccRCC. The LINC02747/miR-608/TFE3 pathway may become a new therapeutic target for ccRCC.With the rapid development of biotechnology, long noncoding RNAs (lncRNAs) have exhibited good application prospects in the treatment of cancer, and they may become new treatment targets for cancer. This study aimed to explore lncRNAs in clear cell renal cell carcinoma (ccRCC). Differentially expressed lncRNAs in 54 pairs of ccRCC tissues and para-carcinoma tissues were analyzed in The Cancer Genome Atlas (TCGA), and the most significant lncRNAs were selected and verified in ccRCC tissues. We found that lncRNA LINC02747 was highly expressed in ccRCC ( Renal cell carcinoma (RCC) is the most common malignant tumor originating from the kidney. According to statistics by Global Cancer 2018, RCC accounts for approximately 2–3% of all malignant tumors in adults . CurrentApproximately 70–90% of the human genome has transcriptional activity, but less than 2% of genes can encode proteins . Long ncThe study was approved by the Ethics Committee of the Third Affiliated Hospital of Suzhou University. All studies were conducted in accordance with the Helsinki Declaration. All patients in studies involving human subjects signed the informed consent forms. Experiments involving animal subjects were performed in accordance with the recommendations detailed in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.https://ibl.mdanderson.org/tanric/_design/basic/main.html). A paired t-test was utilized to identify the differentially expressed lncRNAs between the 54 ccRCC and 54 paired paracarcinoma tissues. Differentially expressed lncRNAs with fold change ≥4 and adj P-value <0.01 were used for the subsequent analysis. The expression of LINC02747 in TCGA and related clinicopathological characteristics of 447 ccRCC patients were analyzed by t-tests. Kaplan–Meier curves were utilized to evaluate the prognostic value of LINC02747 in 447 ccRCC patients. Statistical analysis was performed with R software  and SPSS 22.0 software .TCGA data were downloaded from The Atlas of ncRNAs in Cancer (TANRIC) software , 75% confluent cells were transfected with 50 ng/ml of LINC02747 siRNA, sh-NC, sh-LINC02747, TFE3 specific siRNA, siRNA negative control, miR-NC, miR-608 mimics, miR-608 inhibitor, control, LINC02747, and TFE3 overexpression plasmid. All plasmids were purchased from BoyaBio Co. Ltd. .Cell lines  were purchased from American Type Culture Collection . Dulbecco’s modified eagle medium (DMEM)  with 10% fetal bovine serum (FBS)  was used for the cell culture. After the cells reached the logarithmic growth phase, the concentration was adjusted to 1×10The binding sites of LINC02747, TFE3, and miR-608 were predicted using a database. LINC02747-WT and TFE3-WT luciferase plasmids containing wild-type binding sites and LINC02747-MUT and TFE3-MUT containing mutated binding sites were inserted into the psiCHECK-2 luciferase reporter vector . The luciferase plasmids LINC02747-MUT, LINC02747-MUT, TFE3-WT, and TFE3-MUT were cotransfected with miR-608 mimic into 786-O and Caki-1 cells. After 48 h of culture, double luciferase system was used to detect the fluorescence value of each group.-ΔΔCt method.After transfection for 24 h, total RNA was extracted from cells with the manufacturer’s instructions . Then, the RNA was reverse transcribed into cDNA using the TaKaRa Reverse Transcription Kit  under RNase-free conditions. The primers for the related genes and internal reference were designed and synthesized by Ximao Biotechnology Company , and the primer sequences are shown in Protein was extracted from 786-O and Caki-1 cells after transfection for 48 h. Then, the samples were transferred to a polyvinylidene fluoride (PVDF) membrane, followed by successive incubation with 5% nonfat milk, primary antibodies and secondary antibodies. The relative expression of the target protein was analyzed with GAPDH as a loading control. The primary antibodies anti-TFE3  and anti-GAPDH  were used in this research. After incubation with secondary antibody, the protein bands were imaged using ImageJ software . The experiment was performed three separate times.RIP analysis was performed using an RNA Immunoprecipitation Kit  in accordance with the manufacturer’s instructions, with an antibody against argonaute-2 . IgG served as a control. The purified RNAs were then subjected to qRT-PCR analysis.Cells in the logarithmic growth phase were digested and transferred into a 96-well plate . Cells were gently mixed evenly in the wells. After the cells adhered to the wall, they were transfected in each group for 0–96 h. After treatment, the culture solution was aspirated, and 90 μl of the corresponding complete medium and 10 μl of CCK-8 reagent  were added for incubation in an incubator at 37°C for 1 h. Then, the absorbance value was measured at a wavelength of 450 nm, and the obtained data were statistically analyzed. The relative proliferation rate was calculated as the cell viability at 24, 48, or 72 h/cell viability at 0 h.7 cells per nude mouse) and resuspended in 200 μl of PBS. The BALB/c nude mice  were randomly divided into two groups: one group was injected with control cells, while the other group was injected with 786-O cells with LINC02747 knockdown. First, the axillary skin of the nude mice was disinfected with 1% iodine, and 200 μl of cell suspension was drawn into a disposable microsyringe and injected into the subcutaneous tissues of the axillary skin. Liquid leakage was carefully avoided and the grouping was labeled in detail. The nude mice were observed every 3 days to monitor the growth of the tumor and record the volume of the tumor. The tumor volumes were calculated as 0.5 × (length × width2). After 30 days, the nude mice were sacrificed via cervical dislocation, and the transplanted tumors were removed, weighed, imaged, and stored for subsequent tests.The stably transfected cell lines in the control group in the logarithmic growth phase and the 786-O cell line stably transfected with control or sh-LINC02747 were digested with 0.25% trypsin, centrifuged, mixed with 1 ml of phosphate buffered saline (PBS), resuspended and counted. Then, the cells were prepared  with Tukey’s post-hoc test was used for the comparison between multiple groups, and data at different time points were processed by repeated-measures ANOVA, followed by a Bonferroni post-hoc test. The Kaplan-Meier method with the log-rank test was adopted for survival differences. A value of P <0.05 indicated significant difference.SPSS 22.0  software was used for statistical analysis. The results are expressed as the mean ± standard deviation (mean ± SD). A p <0.01 as the criteria, we found that a total of 20 lncRNAs were upregulated and 329 lncRNAs were downregulated in the ccRCC tissues  was the most obvious. Compared with that in the para-carcinoma tissues, LINC02747 was highly expressed in the cancer tissues (P < 0.001)  and RCC cells . The results revealed that the expression of LINC02747 in the RCC cells was significantly higher than that in the normal kidney cells  (P = 0.004) (P = 0.006) . CCK-8 assay were performed to detect the effect of LINC02747 expression on the proliferation of 786-O cells. The upregulation of LINC02747 significantly promoted the proliferation of RCC cells (P < 0.004) (P < 0.001) (P < 0.01)   and control cells into the subcutaneous tissues of nude mice for 30 days. The growth rate of the tumors in the LINC02747 siRNA group was significantly lower than that in the control group (http://regrna2.mbc.nctu.edu.tw/), we identified the miRNAs  most likely to bind to LINC02747. Then, LINC02747 was overexpressed in 786-O and Caki-1 cell lines, and the expression changes of the above miRNAs were detected by qRT-PCR. The results showed that miR-608 expression was significantly downregulated  (P < 0.01) but did not reduce the luciferase activity of the mutant vector or empty vector (P < 0.001) (P < 0.001) (Nuclear-cytoplasmic fractionation assay demonstrated that LINC02747 was mainly located in the cytoplasm (P < 0.01). In the CCK-8 proliferation assay, we found that upregulation of miR-608 significantly promoted the proliferation of RCC cells (P = 0.023) (P < 0.01) (P < 0.05) . CCK-8 proliferation assay showed that in the cell lines with upregulated miR-608, the upregulation of LINC02747 had no significant impact on the proliferation of RCC cells  and miRDB (http://mirdb.org/). Then, the expression of miR-608 in 786-O and Caki-1 cells was upregulated, and we found that the mRNA expression of TFE3 changed most significantly, and the difference was significant  (P < 0.01) but did not reduce that of the mutant or empty vector . CCK-8 assays showed that upregulation of miR-608 expression had no significant impact on the proliferation of RCC cells when TFE3 was upregulated (To verify whether miR-608 can inhibit the proliferation of RCC by inhibiting TFE3, we transfected 786-O and Caki-1 cells with Control+miR-NC, Control+miR-608 mimic, TFE3+miR-NC, or TFE3+miR-608 mimics. The mRNA and protein levels of TFE3 in the four groups after transfection are shown in in vivo and in vitro experiments confirmed that LINC02747 could promote the proliferation of RCC cells.LncRNAs are the most important type of ncRNA in mammals , and theLncRNAs bind to miRNAs through complementary sequences and competitively downregulate the binding of miRNAs to target mRNAs, thus regulating gene expression, which is one of the main mechanisms of posttranscriptional regulation . In thisIn conclusion, it was confirmed by analyzing the relevant data in TCGA and the qRT-PCR results of clinical specimens that lncRNA LINC02747 is highly expressed in ccRCC and is closely related to the high TNM stage and histological grade and the poor prognosis of patients. LINC02747 upregulates the expression of TFE3 by adsorbing miR-608, ultimately promoting the proliferation of RCC cells. The above findings indicate that LINC02747 acts as an oncogene in ccRCC and may be developed as a molecular marker for the diagnosis and prognosis of ccRCC. The LINC02747/miR-608/TFE3 pathway may become a new therapeutic target for ccRCC.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Third Affiliated Hospital of Soochow University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Third Affiliated Hospital of Soochow University.XJ and XH conceived and designed the study and helped to draft the manuscript. YS, FZ, and XW performed the data collection. ZW performed the statistical analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Schneiderian membrane (SM) perforation is a major complication of maxillary sinus elevation with simultaneous bone grafting, yet under this scenario there is no standard biomaterial that maximizes favorable tissue healing and osteogenic effects.To compare the effect of advanced platelet-rich fibrin (A-PRF) and collagen membrane (CM) on a perforated SM with simultaneous bone grafting in a maxillary sinus elevation model.n = 12), (ii) group A-PRF: A-PRF and DBBM (n = 12). Radiographic and histological evaluations were performed at 1 and 4 weeks post-operation.After perforation of the SM was established, 24 animals were randomly divided into two groups: (i) group CM: CM and deproteinized bovine bone mineral (DBBM) (p < 0.0001). At 4 weeks, the osteogenic pattern was shown as from the periphery to the center of the sinus cavity in group A-PRF.At 1 week, an intact SM was found in group A-PRF. At each time point, the number of inflammatory cells at the perforated site was higher in group CM, and the area of new osteoid formation was significantly greater in group A-PRF (The higher elasticity, matching degradability, and plentiful growth factors of A-PRF resulted in a fully repaired SM, which later ensured the two osteogenic sources from the SM to generate significant new bone formation. Thus, A-PRF can be considered to be a useful bioactive tissue-healing biomaterial for SM perforation with simultaneous bone grafting. Long-term loss of the maxillary posterior teeth often leads to a series of complications, such as maxillary sinus pneumatization and ridge atrophy. These conditions may increase the implant failure rate . To manaHowever, despite accurate preoperative radiographic investigations and surgical maneuvers, Schneiderian membrane (SM) perforation can occur during the elevation process, and it has a reported incidence of 56% . IrregulThe treatment of the perforation during sinus elevation depends on the perforation size. When the perforation is less than 5 mm , the mosNevertheless, the dense structure of the CM might block the osteogenesis of the SM. The SM is a possible source of osteogenesis in the maxillary sinus , and thePlatelet-rich fibrin (PRF), as a self-clotted preparation of platelet-concentrated and autologous blood–derived biomaterial, has been advocated in several studies and produced favorable outcomes for SM perforation during maxillary sinus floor elevation . AdvanceAlthough an animal study  has demog for 14 min. Then, three layers emerged in the anticoagulant-free tube. Acellular plasma was separated, and the red blood cells attached to the A-PRF were removed with a knife   with an accelerating voltage of 15 kV.The compressed A-PRF, moist CM, and the natural SM were selected for mechanical testing. All membranes were cut into 20 × 5-mm (length × width) rectangular strips for mechanical testing of elasticity. The mechanical properties of different membranes were measured at a stretching speed of 1 mm/min with a desktop universal testing machine , where the maximum load cell capacity was 100 N under standard ambient conditions at 25°C ± 3°C and 50 ± 25% relative humidity (RH). The elastic modulus was defined as the average slope of the initial part (0–10% strain) of the stress–strain curve.n = 12), (ii) group A-PRF: A-PRF and DBBM (n = 12). The effect of each group  was assessed at two healing time points that were 1 and 4 weeks post-operation.Animal experiment protocols were approved by the Animal Ethics Committee of Chongqing Medical University (CQHS-IRB-2018-07) and were conducted according to National Institutes of Health guidelines. We used 24 male New Zealand rabbits with weights ranging from 3 to 3.5 kg. After the SM perforation was established, rabbits were randomly divided into two groups: (i) group CM: CM and deproteinized bovine bone mineral (DBBM) , calcein , and alizarin complexone  at the first, second, and third week post-operation, respectively, to observe the osteogenic patterns.The rabbits were monitored, and antibiotics and analgesics were administered on the first 3 d post-operation.At the 1- and 4-week healing time points, rabbits were euthanized by injection of sodium pentobarbital  through the central ear artery. The maxillary sinus samples were collected and processed for micro–computed tomographic (micro-CT) analysis and histological evaluation.The 4-week post-operation maxillary sinus samples were analyzed using a micro-CT . The scanning condition was acquired at a resolution 14.91 μm (130 kV and 60 μA). Three-dimensional reconstruction of the interest areas was performed withμCT80 .Half of the specimens in the 4-week groups were obtained and dehydrated in a graded series of ethanol and embedded in methyl methacrylate . Subsequently, the specimens were prepared using the Hard Tissue Sawing System . Tissue slices were observed with a fluorescent microscope  for fluorescent labeling. Finally, the samples were stained with Van Gieson (VG). In addition, the 12 specimens at 1 and 4 weeks post-operation were decalcified and embedded in paraffin. They were then stained with Picro-Sirius red stain for observation of re-epithelialization during SM repair. Hematoxylin-eosin (H&E) staining was used to evaluate early and later inflammatory responses. Aniline blue and osteocalcin immunohistochemical staining (IHC) were also used to assess formation of new bone. Osteoclast activity was observed using tartrate-resistant acid phosphatase (TRAP) staining.The tissue sections showed that the fracture of the basal bone corresponded to the perforated area of the SM , and the•Relative proportion of different cells (%): the percentage of pixels  in the ROIa according to H&E staining.•The area of new osteoid formation (%): blue-stained mineralized tissue area including osteocytes with the use of aniline blue staining, which is described as a percentage of the whole ROI for semiquantitative analysis .•Relative expression of osteocalcin (%): percentage of pixels associated with deeply stained osteocalcin-positive cells in the ROIa by IHC staining.•The relative expression of osteoclasts (%): osteoclasts are recognized as the TRAP-positive cells with the use of TRAP staining sections, which are expressed as a percentage of the ROI for semiquantitative analysis .t-tests using GraphPad Software v6 , and statistical significance was considered at p < 0.05. All data are expressed as the mean ± standard deviation.All data had a normal distribution and were analyzed using SPSS Statistics version 20.0 . Statistical analysis was performed by Student All experimental animals undergoing surgical procedures maintained a healthy status throughout the entire experimental period. No complications were observed during the postoperative period.Microstructures of CM and A-PRF were examined by SEM. As shown in p < 0.0001), indicating that A-PRF had superior elasticity.Tensile properties among CM, A-PRF, and the natural SM are shown in the stress–strain plot . The yieA dome-shaped space was observed in the elevated maxillary sinus in two groups . The DBBH&E staining  revealedMicro-CT  reconstrAs shown by Picro-Sirius red staining at 4 weeks post-operation , the CM Based on aniline blue staining , there wIn IHC, group A-PRF exhibited strongly osteocalcin-positive cells under the SM, whereas almost no positive expression was shown in group CM . In TRAPWhile distinguishing the osteogenic patterns in each group, a fluorochrome label of new bone mineralization was detected. Weak fluorescence signals were observed in group CM, whereas group A-PRF displayed stronger fluorescence intensity . The flup < 0.001; 33.87 ± 1.29% vs. 12.23 ± 0.25% at 4 weeks, p < 0.0001). The relative proportion of fibroblasts in group A-PRF was measured compared to group CM, which was significant . Eosinophils in group A-PRF were significantly fewer than in group CM   .p < 0.0001; 0.62 ± 0.06% vs. 0.09 ± 0.01% at 4 weeks, p < 0.001) (p < 0.001). In addition, there was a significant difference between group A-PRF and group CM in the relative expression of osteoclasts   . The rel < 0.01) .Perforation of the SM occurs in the maxillary sinus elevation at a frequency of 10–56% . Althougp < 0.0001), suggesting that the superior elasticity shown in A-PRF was due to its reticular and porous microstructure. Therefore, the superior elasticity of A-PRF avoided secondary perforation caused by breathing movement and overfilling of DBBM in the repair site, which provided a good foundation for perforated SM repair.A CM is a double-layered absorbable barrier membrane that has been widely used in guided bone/tissue regeneration (GBR/GTR) . The bacin vivo, timely degradation and appropriate immunogenic characteristics are important prerequisites to facilitate the repair process. As shown in p < 0.001), whereas the fibroblasts were greatly increased compared to group CM at an early stage   . After i process , and thi process . Specifi process . Additio process . Therefo process , and an  process . OverallSM repair restores the integrity of the SM and also establishes a microenvironment suitable for new bone formation and remodeling after the maxillary sinus floor elevation process. Based on previous studies, there are two sources of osteogenesis in the elevated sinus floor area. One is osteogenesis from the basal bone, and the other is from the SM . FiguresThis study revealed that (i) significant SM repair occurred when utilizing A-PRF, and the degradation of A-PRF was matched with the SM repair process at an early stage; (ii) bone remodeling in the sinus cavity was active, and a greater amount of new bone formation occurred under the perforated SM area in the A-PRF group at a later time point. This is the first preclinical study evaluating A-PRF as an alternative to CM for repair of SM perforation with the filling of DBBM simultaneously.Several clinical studies investigated a potential benefit due to the placement of a blood product membrane to the perforated SM . Those cThe rabbit experimental model was first introduced by The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Animal Ethics Committee of Chongqing Medical University (CQHS-IRB-2018-07).LX: design, methodology, data analysis, drafting article, validation, animal experiments, and data collection. SY: critical revision of article, statistics, and approval of article. ZM: responsible for the assistance of animal experiments and methodology. DL: responsible for micro-CT and H&E staining. JS: funding acquisition, supervision, conceptualization, and formal analysis. TC: conceptualization, project administration, funding acquisition, methodology, and writing-review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Since the role of hyperlipidaemia in the pathogenesis of AAA remains controversial, we aimed to evaluate the significance of HO-1 in the development and progression of AAA in normolipidemic animals. The experiments were performed in HO-1-deficient mice and their wild-type counterparts. We demonstrated in non-hypercholesterolemic mice that the high-dose of AngII leads to the efficient formation of AAA, which is attenuated by HO-1 deficiency. Yet, if formed, they are significantly more prone to rupture upon HO-1 shortage. Differential susceptibility to AAA formation does not rely on enhanced inflammatory response or oxidative stress. AAA-resistant mice are characterized by an increase in regulators of aortic remodeling and angiotensin receptor-2 expression, significant medial thickening, and delayed blood pressure elevation in response to AngII. To conclude, we unveil a dual role of HO-1 deficiency in AAA in normolipidemic mice, where it protects against AAA development, but exacerbates the state of formed AAA.Abdominal aortic aneurysm (AAA) bears a high risk of rupture and sudden death of the patient. The pathogenic mechanisms of AAA remain elusive, and surgical intervention represents the only treatment option. Heme oxygenase-1 (HO-1), a heme degrading enzyme, is induced in AAA, both in mice and humans. HO-1 was reported to mitigate AAA development in an angiotensin II (AngII)-induced model of AAA in hyperlipidemic ApoE Abdominal aortic aneurysm (AAA) is one of the most prevalent age-related cardiovascular disorders. It is a local at least 50% dilatation of the abdominal aorta occurring in the infrarenal area. Due to its asymptomatic development and progression, AAA is often overlooked and diagnosed late, bearing a high risk of rupture and sudden death . BesidesFormation of AAA is accompanied by extensive activation of proteolysis, high inflammation, oxidative stress, which lead to smooth muscle cells death and aortic wall rearrangement. However, the detailed activation pathways are still not well elucidated . One of HMOX1 gene) is an inducible rate-limiting enzyme, degrading heme into ferrous ions, carbon monoxide, and biliverdin. HO-1 activity can be altered by porphyrins, and the species of cation of metal linked to porphyrin rings determines the direction of the change. Those modulators of HO-1 activity, together with knockout models, were used throughout years to demonstrate high antioxidant, anti-inflammatory, and antiapoptotic properties of its metabolites in numerous models [HMOX1 gene promoter polymorphism affects the expression of HO-1 in humans, suggesting an additional indication for the clinical relevance of studies on the role of HO-1 in AAA pathophysiology [Heme oxygenase-1 . In addysiology .-/- mice and porcine elastase model [Up to date, the role of HO-1 in AAA has not been fully elucidated as the available reports draw contrasting conclusions. Some of them indicate for detrimental role of HO-1. It is upregulated in human AAA , and we se model . Moreovese model ,15,16. T−/− mice and HO-1+/+ littermates both on mixed C57/Bl6 × FVB background. Six-month-old male mice of verified genotype were used . The genotype was verified prior to the experiment by DNA analysis. The animals were maintained under specific pathogen-free conditions in the individually ventilated cages (14/10 h light/dark cycle at a temperature of 22 ± 2 °C) and were provided with a 25% fat diet and water ad libitum. All experimental procedures were approved by the Second Local Ethics Committee for Animal Experiments in Krakow (No. 74/2016 and 110/2016) and performed in accordance with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.Experiments were performed on HO-1Mice were infused via osmotic minipumps  with angiotensin II  or saline (sham group) for 28 days. Osmotic pumps were placed subcutaneously under Avetin (TBE) anaesthesia  as described previously . The folAll mice underwent non-invasive blood pressure measurement by tail-cuff plethysmography  which was preceded by a period of adaptation. The measurements were performed by one person to decrease any bias. The results were presented as a percentage change compared to day 0 (after adaptation time and before the administration of any drugs). This way of data presentation was chosen to plot the impact of administered compounds better and to minimize the influence of phenotypic and age-related changes between littermates. Importantly, no difference in mean blood pressure between genotypes was observed at day 0.Changes in the abdominal aorta diameter were monitored using the high-frequency ultrasound imaging system . Two-dimensional (B-mode) imaging using a 22–55 MHz linear-array transducer (MS550D) synchronized to the electrocardiographic signal was done. The animals were placed in a supine position on a heated table under inhalation anaesthesia with isoflurane (1.5–2%). The abdominal cavity was shaved, and a pre-warmed ultrasound gel was applied to the area of interest. Longitudinal images of the suprarenal and infrarenal aorta and transverse images at the level of the abdominal aorta between the diaphragm and the outlet of the left renal artery were acquired in the B-mode and M-mode to assess the maximum cross-sectional diameter (during diastole) and aortic area in real-time for each mouse at each time point. The detailed analysis scheme is presented in .Right after euthanasia, approximately 1 mL of blood was collected from the retroorbital sinus to a tube coated with EDTA. Blood cell count was analyzed using ABC Vet Hematology Analyzer .® Green PCR Master Mix according to the manufacturer’s instructions . Primer sequences are gathered in eEF2) was used as a reference gene. Relative gene expression was calculated using the ΔΔCt method.RNA from 3 mm fragment of abdominal aortic tissue was extracted with the RNeasy Mini Kit  according to the manufacturer’s instructions. cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit . RT-qPCR was conducted on Step-One Plus Real-Time PCR Systems using a Power SYBRHaematoxylin and eosin stainings were performed on 9 µm tissue slices using the method available on the IHCworld protocols website. Trichrome staining was done on 9 µm tissue slices using the Trichrome Stain (Masson) Kit . Samples were analyzed under a light microscope  with NIS elements BR software  at magnifications of 100× and 400×.Immunofluorescent stainings were done in frozen 10 µm , or 30 µm (immune cells) specimens of the abdominal aorta. Samples were fixed with methanol or ice-cold acetone and blocked in 10% donkey serum with 0.05% Tween-20 for 1 h at room temperature (RT). After washing in PBS, samples were incubated overnight (4 °C) with antibodies: anti-HO-1 , anti-collagen I or collagen III , anti-osteopontin . On the next day, samples were washed and incubated with secondary donkey antibodies conjugated with either Alexa Fluor 488 or Alexa Fluor 568  for 1 h at RT. Nuclei were counterstained with DAPI . For immune cell detection, PE-conjugated antibodies were used: anti-CD45 , anti-CD86 , anti-CD163 . Samples were analyzed under a meta laser scanning confocal microscope  and analyzed using ImageJ software (Wayne-Rasband (NIH)).Intracellular heme concentration was checked using a fluorescence-based assay. First, 3 mm part of abdominal aorta was rubbed and resuspended in 2 M oxalic acid . Half of the sample volume was heated at 95 °C for 30 min leading to iron removal from heme. Fluorescence (excitation 400 nm– emission 662 nm) of the resultant protoporphyrin was assessed by Tecan Spectra II Microplate Reader . The endogenous protoporphyrin content  was subtracted. Data were normalized to total protein concentration in each sample.2, 0.2 mM NaN3, 50 mM Tris-HCl, pH 7.6) at 37 °C in a dark wet chamber for 2 h. Next, slides were washed in MilliQ water, fixed for 1 min in ice-cold acetone, and blocked in the wet, dark chamber with 10% GS with 0.05% Tween-20 for 1 h at RT. Further, scraps were overlaid with MMP-2 and MMP-9 antibodies  in 1% GS with 0.05% Tween-20 and left overnight at 4 °C in a dark wet chamber. On the next day, samples were washed and incubated with anti-rabbit antibodies conjugated with Alexa Fluor 568 . Nuclei were counterstained with DAPI . Samples were mounted in DAKO mounting medium and analyzed under a meta-scanning confocal microscope LSM 880  at magnification 400× and analyzed using ImageJ software.In situ gelatinolytic activity in the media of frozen abdominal aorta was performed as described previously . Slides 2 atmosphere. Transfections of HASMC were performed using 20 nM siRNA targeted against human HMOX1 (s11056) or scrambled siRNA  using Lipofectamine™ RNAiMAX Transfection Reagent  in Opti-MEM I Reduced Serum medium . At 48 h after transfection, the cells were incubated for 24 h with 50 nM AngII . For proliferation assessment, cells were washed with PBS, fixed with ice-cold methanol, blocked in 3% BSA, and incubated with anti-PCNA antibody  at 4 °C overnight. Next day, cells were incubated with secondary antibody conjugated with Alexa Fluor 568  and Hoechst 33342 to stain the nuclei.Human aortic smooth muscle cells (HASMC) were grown in Medium 231  supplemented with 10% FBS and 1% penicillin streptomycin. Cells were cultured at 37 °C in a humidified incubator in 5% COp < 0.05), which were not included in the statistical analysis of the results (GraphPad Prism 8 software). p < 0.05 was accepted as statistically significant.Data are presented as mean ± SEM. Two-way ANOVA, followed by Tukey’s post hoc test, was used for comparison of more than two groups. Fisher’s exact test was used to calculate the frequency of aneurysm appearance and rupture. Mann-Whitney test was used for comparison of 2 groups. Grubbs’ test was used to detect statistically significant outliers (p < 0.05), which led to the formation of AAA in 30–40% of mice, as reported by several groups, including us [The commonly used dose of angiotensin II (AngII) in AAA-related studies is 1000 ng/kg/min; however, such a quantity leads to inefficient AAA formation in wild-type animals. Thus, in our study, we used the standard  and high dose  of AngII. Mice were kept on a fat-enriched diet, containing 25% saturated fat. Such a regime did not alter the plasma cholesterol and triglycerides level A,B, impluding us   to develop AAAs less frequently in comparison with wild-type animals , HO-1 ip = 0.03). Moreover, total heme content was 3–5 times higher in mice that developed the aneurysm, irrespectively of genotype . On the contrary, aortic heme content was higher (p = 0.028) in saline-infused HO-1-deficient animals but was not significantly increased upon AngII infusion (p = 0.3). The analysis of the expression of ALAS1, FLCVR1, and SLC40A1 (ferroportin) revealed no significant differences (ALAS1) is visible in the saline-treated HO-1 KO mice.The obtained results showed that AngII significantly increases heme content in the aortic wall  tended to rise in WT animals upon AngII infusion.Then, we investigated the level of collagen regulators. The expression of egulated A,B. In HSPP1 gene) was increased both at mRNA and protein level. The induction was statistically significant in both genotypes, but much more pronounced in WT animals  and their inhibitors (TIMPs) . We foun9 levels A, known TIMP1 by AngII was stronger in WT than in HO-1 KO animals. In saline-treated KO mice, TIMP2 tended to be higher in comparison to WT counterparts. The difference was significantly heightened upon AngII infusion. The level of TIMP3 remained unaltered  and co-stained samples for MMP2 and MMP9. AngII enhanced gelatinases activity. Whereas. in formed AAA. it spread almost through the whole aneurysmal tissue; in the unexpanded aortas, it was rather site restricted. The signal for MMP2, but not MMP9 colocalized with gelatinases activity in HO-1-deficient animals. In WT mice, there were small areas of colocalization only with MMP2 B. The exnaltered C.The infusion of AngII to normolipidemic C57Bl6 mice as a model of AAA was introduced around 20 years ago ,26; yet,The impact of HO-1 deficiency has already been studied in two murine models, characterized by high AAA incidence: porcine elastase infusion  and AngII infusion to hyperlipidemic ApoE KO mice (80% formation of AAA) ,12,27. BIn our study, we decided to use non-hyperlipidemic animals to infer about the disease susceptibility. We show a dual role of HO-1 deficiency, which, on the one hand, protects against AAA development, but still, on the other, may aggravate the already formed aneurysms and promote their rupture. Interestingly, such seemingly irreconcilable results between mice strains have been previously reported in the case of the use of anti-TGFβ antibodies ,32. FurtIn the previous reports, HO-1 deficiency-induced exacerbation of AAA formation was associated with enhanced inflammatory response, macrophage infiltration, and oxidative stress ,12. ThusHMOX1 results mainly from the infiltration of immune cells to the aortic wall and not from its upregulation in regular aortic cellular components. The previous data were obtained in the models, in which the infiltration of immune cells is facilitated. In the elastase mode, there is an artificial imposition of the aortic rearrangement and the induction of vascular damage. Such a procedure may accelerate immune cell homing to the aortic wall and aortic rearrangement, and thus impede the verification of the possible mechanisms underlying altered susceptibility to AAA formation. Accordingly, the ApoE KO mice have much more immune cells, including macrophages, in the aorta in comparison to WT mice [Undoubtedly, infiltration of immune cells to the aortic wall is the prerequisite of the remodeling and loss of medial smooth muscle cells ,34. Our  WT mice ,36, whicAT2R expression, which inversely correlates with blood pressure values [Ang II concentration in plasma in physiological state is around 50 fmol/mL and rises to 100 fmol/mL in hypertension. The kidney levels are approximately 2–3 times higher . The come values . FinallyIntraperitoneal injections of heme were shown to decrease AAA development, and the protective effect was attributed to HO-1 induction . Here, wLiterature data clearly state that the mechanical resistance of the vessel wall is highly reliant on the collagen network. The progression of AAA and its susceptibility to rupture is contingent on fine-tuned collagen turnover. In the aortic wall, the load-bearing proteins are proteolysis-resistant fibrillar collagens type I and III ,42. HereMYH11 and ACTA2 mutations [During the study, we noticed a significant AngII-induced thickening of media upon AngII in HO-1 KO mice. It was mostly caused by the different orientation of SMCs. Instead of being parallel, SMCs were perpendicular to the lumen. In all the animals, they were localized between the 2nd and 3rd elastin layer; however, in some mice, they were extended throughout the whole vessel wall. Such disarray has already been observed in thoracic aneurysm patients with utations .TIMP2 level [The aortic SMCs in AngII-infused HO-1 KO mice were not in close contact with each other; instead, there were separated by acellular spaces. Presence of acellular areas in aneurysms is well known as idiopathic medial cystic degeneration or mucoid ECM accumulation . In geneP2 level .MMP3 and TIMP1 level, we suppose that these may be the key players in the aortic remodeling upon high-dose AngII. Notably, MMP3 activity is essential for the development of AngII-induced aneurysms [MMP2 and MMP9 are known to be pivotal in human aneurysm development . In thisneurysms , and it PAI. It stays in line with previous data reporting an inverse association between HO-1 expression and PAI induction in the injured aortic walls [Undeniably, the infusion of AngII in HO-1-deficient animals induced some aneurysmal features, such as the presence of acellular spaces, increased MMP2 expression, and altered collagen content. Still, despite those characteristics, the aortic diameter remained unchanged. It may indicate for a presence of a ‘switch’ which keeps the aorta restrained, preventing its expansion. In our study, we observed a differential and prominent induction of ic walls . Interesic walls . Thus, iic walls . Furtheric walls ,57. Impoic walls .+ macrophages, which infiltrate aneurysmal tissue and highly express HO-1, and which can disappear in HO-1 KO mice [This research has several limitations. To increase AAA incidence, we used a high dose of AngII, which had not been regularly used in previous studies. It would be essential to perform additional experiments to define this model and outline any possible unspecific side-effects. Secondly, we used mice having the global knockout of HO-1. Identification of exact cell population, which is responsible for the observed phenomenon, would be of utmost importance. Especially, the role of CD163 KO mice ,60, shouPAI, TIMP2, and AT2R expression, significant medial thickening, and delayed blood pressure elevation in response to AngII. The evidenced dual role of HO-1 may result from multiplicitous events associated with AAA formation. Whereas, during the initial steps, HO-1 abundant macrophages trigger the remodeling of the aortic wall, during the further steps, they may be also responsible for the proper disposal of harmful molecules ensuring AAA stability. Undoubtedly, the role of HO-1 is equivocal and shall be addressed in more detail during every step of AAA initiation and stability.To conclude, high-dose of angiotensin II leads to efficient formation of AAA in non-hypercholesterolemic mice, which is attenuated by HO-1 deficiency. Yet, if AAA is formed, it is significantly more prone to rupture upon HO-1 shortage. Differential susceptibility to AAA formation is not dependent on inflammatory response or oxidative stress induction. AAA-resistant mice are also characterized by enhanced


Under these conditions, the perovskite surface was preserved from interaction with the solvent. Over a timescale of 90 days, HP-PSCs were systematically compared to reference cells with carbon-based top electrodes deposited by doctor blading (DB). We found that all the photovoltaic parameters recorded in HP-PSCs during time under ambient conditions settled on values systematically higher than those measured in the reference DB-PSCs, with efficiency stabilized at around 6% within the first few measurements. On the other hand, in DB-PSCs, a long-lasting (~14 days) degrading transient of the performances was observed, with a loss of efficiency from an initial ~8% to ~3%. Moreover, in HP-PSCs, a systematic day-by-day recovery of the efficiency after operation was observed (Δ~2%) by leaving the cell under open circuit, a nitrogen environment, and dark conditions. Noteworthily, a full recovery of all the parameters was observed at the end of the experiment, while DB-PSCs showed only a partial recovery under the same conditions. Hence, the complete release of solvent from the carbon contact, before an interface is established with the perovskite layer, offers a definite advantage through the long period of operation in preventing irreversible degradation. Our findings indeed highlight the crucial role of the interfaces and their feasible preservation under nitrogen atmosphere.Carbon-based top electrodes for hole-transporting-layer-free perovskite solar cells (PSCs) were made by hot press (HP) transfer of a free-standing carbon-aluminum foil at 100°C and at a pressure of 0.1 MPa on a methylammonium lead iodide (MAPbI Perovskite solar cells (PSCs) based on organometal halide perovskite materials have shown a rapid development since their birth in 2009, when Miyasaka et al. . In a different approach and to reduce the device cost, carbon contacts were made directly on the perovskite layer by hot pressing  , PbI2 (main peak at 2θ = 12.7°) or graphite (main peak at 2θ = 26.55°). On the basis of this spatial-resolved investigation, the line profiles of MAPbI3, PbI2, and graphite  are traced as shown in 2 amount inside and outside the contact area is almost negligible meaning that no macroscopic degradation of the perovskite layer occurred during contact formation.The two methods used to fabricate carbon electrodes by doctor blading (DB) and hot pressing (HP) on twin MAPbIoc). The best cell works at an efficiency of 7.94% with short-circuit photocurrent density (Jsc) = 14.73 mA/cm2, open-circuit voltage (Voc) = 1.01 V, and fill factor (FF) = 53.35%. These values closely match the data reported in the literature for similar device architectures and commercial C-based pastes, as summarized in Cai et al. . This finding can be equally interpreted as an improvement of the perovskite lattice quality during time or as a reduction of the PbI2 amount (by a factor 0.6 from day 14 to day 90 in the HP-PSC) that would imply the overall mass (including the organic cations) preserved during the cycle of operation. The measured lattice improvement cannot be fully ascribed to compositional disuniformities along the sampled area, since this would produce smaller normalized variations in the PbI2/MAPbI3 peak intensity ratio (~0.1–0.2).The J-V curves at the beginning and end of operation, together with the curves taken at day 14, testify that a recovery of the performances can be achieved by both DB-PSCs and HP-PSCs . It is nIn conjunction with a recovery of the active material structure that was observed in both samples, the uncomplete restoring of the photovoltaic parameters in the DB-PSC with respect to what occurs in the HP-PSCs at the end of the cycle (day 90) addresses that the C/perovskite interface has been irreversibly modified.oc, Jsc, and FF . This is clearly stated by HP-PSCs show a different behavior. As a piece of first evidence, the device does not exhibit a marked transience in the PCE values after initial operation . Moreoveoc, Jsc and FF  migration of ions in the perovskite layer under illumination and electric field , (2) defects formation under operation and self-curing of the perovskite , including indeed all data along the degradation-recovery path, are summarized as box plots in The statistical distributions of the VLow temperature preparation of Carbon-based top-electrodes were done by hot press transfer method of a free-standing C+Al foil at 100°C and pressure of 0.1 MPa and integrated in PSCs architectures. HP-PSCs were systematically investigated during time by acquiring J-V curves in ambient conditions. After operation, the cells were left under open circuit voltage, nitrogen environment, and dark conditions. Limited deterioration and significant day-by-day recovery were observed along the entire timescale, which suggests the carbon/perovskite interface is sufficiently preserved from degradation during operation, as well as a capability for self- and nitrogen-assisted healing of the perovskite layer while not operating. This capability is definitely demonstrated by the full recovery of all the device parameters, primarily the efficiency, at the end of the experiment. On the opposite, the reference DB-PSC exhibited a monotonic descending trend of the performances during time that has drawn a transient behavior driven by degradation. The action of solvent during contact formation and its possible further action during time is indeed demonstrated to have deleterious impact on the cell performances in the long period. Solvent-based deposition methods for the C-contact indeed demand tailored procedures to mitigate the negative effects. We additionally argue that, above the full recovery of the HP-PSCs performances, a further increase of the efficiency would be feasible by improving the selectivity of the C-contact and the conductivity of the overall top-electrode.3 layer was deposited by solution processing on a 90 nm-thick meso-porous-TiO2 on compact-TiO2/FTO. Before preparation of perovskite film, a perovskite precursor solution and the substrates were warmed at 70°C. A 1.5 M solution of methylammonium iodide (MAI) and PbI2 (1: 1 in mol) in N, N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO) (4:1 v/v) was poured onto the TiO2 mesoporous substrate and spin-coated . The as-prepared film was pot-roast vapor annealing (PR-VA)-treated at 100°C for 10 min on a hot plate. The resulting perovskite layer was compact and uniform. The size of the grains was in the range 400–800 nm.A 450 nm-thick MAPbIA = 0.25 cm2) and thickness (30 μm) of the top electrode. Then, the PSC with the wet carbon film was dried at 120°C for 15 min (according to the Dyenamo datasheet) in nitrogen atmosphere to remove the solvent and to promote the curing of the paste. Trials by using 100°C annealing produced similar performances in devices.To complete PSC architecture, a carbon layer was deposited following two methods. In the doctor blading method, a commercial carbon paste (Dyenamo DN-CP01) was coated onto the perovskite layer with a blade. A kapton tape frame is used to define the area (A = 0.25 cm2) was formed and directly hot pressed onto the perovskite layer under the pressure of 0.1 MPa at 100°C for 40 min.In the hot press transfer method, glass substrate was coated with the commercial carbon paste by doctor blading, forming a wet carbon film. Then it was soaked into ethanol for a solvent-exchange. After that, the carbon film was peeled off from the glass substrate and, differently from ref  from Peccell PEC-L01. The solar cells devices were masked (shadow mask) to define the active area of 0.25 cm2 and then irradiated from the backside at the FTO/TiO2 side. Scan range was from −0.1 to 1.2 V for forward scan and from 1.2 to −0.1 V for reverse scan with a step of 0.01 V. The delay time was of 0.1 s.J-V characteristics were measured by a digital source meter (Keithley model 2401) under AM 1.5-simulated sunlight (100 mW/cmThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.2/perovskite bilayers. ID, AL, AG, and TM made supervision actions. AA conceived the experiment, coordinated the activities, and refined the paper.SV deposited the C-based contact, performed the electrical characterization of the devices, and wrote the paper draft. ES performed the X-ray characterization of the devices. SS prepared the free-standing C-foils. YN and AJ prepared the TiOThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To determine the level of Escherichia coli (E. coli) contamination, after induction with 150 µL of breast milk, the B-Per bacterial protein extraction kit was added to a solution containing an alginate-based microcapsule assay. Within this 3 mm spherical novel sensor design, X-Gal  was entrapped at a concentration of 2 mg/mL. The outward diffusing X-Gal was cleaved by β-galactosidase from E. coli and dimerized in the solution to yield a blue color after incubation at 40 °C. Color intensity was correlated with the level of E. coli contamination using a categorical scale. After an 8 h incubation period, a continuous imaging scale based on intensity normalization was used to determine a binary lower limit of detection (LOD), which corresponded to 102 colony forming unit per mL (CFU/mL) and above. The cost of the overall assay was estimated to be $0.81 per sample, well under the $3 benchmark for state-of-the-art immune-based test kits for pathogen detection in biofluids. Considering the reported binary LOD cutoff of 102 CFU/mL and above, this proposed hydrogel-based assay is suited to meet global requirements for screening breast milk or milk for pathogenic organisms of 104 CFU/mL, with a percentage of false positives to be determined in future efforts.There are no existing affordable diagnostics for sensitive, rapid, and on-site detection of pathogens in milk. To this end, an on-site colorimetric-based sustainable assay has been developed and optimized using an L Breast milk is widely considered to be the gold standard for infant nutrition, offering essential nutrients and antibodies that enhance an infant’s health outcomes ,2. BreasEscherichia coli (E. coli) in food samples [d-Galactopyranoside). For E. coli in breast milk, the lower limit of detection (LOD) was reported to be 105 colony forming unit per mL (CFU/mL) [Papers or bioactive paper assays for bacterial detection in dairy milk and orange juice exist in a simple kit that would allow untrained personnel to carry out sensitive, multiplexed detection of  samples ,25. Thes(CFU/mL)  for a tw(CFU/mL) .5E. coli), rapid (with assay development times between two and eight hours), portable , environmentally friendly (no solvents and biodegradable), low-cost and requiring minimal training was developed. In order to circumvent contaminant entrapment, a simple assay design based on radial diffusion of an entrapped X-Gal substrate from an alginate microcapsule was proposed. The mass transfer component of the assay was comprised of a cross-linked alginate membrane with an established molecular weight cutoff (MWCO) of 70 kDa [d-mannuronic and -α-l-guluronic acid residues, is a widely used biomaterial in tissue engineering and regenerative medicine [Motivated by these observations, a bioactive assay for detection of pathogens that is sensitive  protocol, was used for all testing described here. SCU-104 is capable of metabolizing lactose and shows regulatory behavior with regard to the lac operon that conforms to classic models. Luria Bertani broth  powder, N,N-Dimethylformamide , medium-viscosity alginic acid , and β-galactosidase  were purchased from Sigma-Aldrich . The B-PER Direct Bacterial Protein Extraction Kit  and X-Gal  were procured from Thermo Fisher Scientific . All other reagent grade chemicals were provided by the Bioengineering Department at Santa Clara University, Santa Clara, California, USA).E. coli strain SCU-104 was cultured in 2 mL of 2.5% (w/v) autoclaved LB broth. Optical density (OD: 600nm) was monitored hourly using a Genesis 10S UV-Vis spectrophotometer  [8 cells/mL) were harvested by centrifugation at 3000 rpm for 5 min. The bacterial cell pellet was resuspended in 200 µL of 50 mM sterile sodium phosphate buffer (pH = 7.4). A range of bacterial cell concentrations (3.16 × 105–1.00 × 107 CFU/mL) was prepared in order to construct a scaled detection ladder for optimization experiments. For the lower LOD determination, a range of 0–1.00 × 108 (CFU/mL) was used post induction. Two separate batches of bacteria but a single batch of breast milk were used for the above-stated optimization and LOD determination experiments.MA, USA) . During E. coli cells were lysed in order to access intracellular β-galactosidase protein according to the B-PER extraction protocol [v/v) lysozyme, 1–5% (v/v) DNAase, and 90–98% (v/v) B-PER) was added to E. coli. The solutions were then vortexed and incubated at 37 °C for lysing times ranging from 10 to 25 min. A primary positive control for enzyme induction comprised of 1 mg of X-Gal powder was added to the lysed solution corresponding to the highest concentration in the bacterial ladder (1.00 × 107 CFU/mL), followed by incubation at 37 °C for 20 min. A visible color change to blue was an indicator of β-galactosidase expression. protocol . Two hunw/v) solution of alginate was prepared using 0.9% (w/v) NaCl as a solvent and autoclaved. X-Gal volumes ranging from 25 to 100 µL aliquoted from a stock solution of 20 mg X-Gal/DMF  were dissolved in 1 mL of alginate and stirred for 4 h. Alginate gel assay fabrication was based on the principle of ionotropic gelation [2 solution for cross-linking. After 30 min, the resultant hydrogel capsules were washed three times with 0.9% NaCl to stop cross-linking. Shown in gelation . The bioβ-galactosidase, which was the basis of the colorimetric assay. Post induction with breast milk, the lysed solutions of the bacterial dilution ladder were used to simulate contaminated samples at the milk bank, after which a single microcapsule containing the X-Gal substrate was added to the mixture. As X-Gal diffused out of the microcapsule, it was cleaved by β-galactosidase, capsule burst as a result of oncotic pressure was a binary indicator of protein expression. Due to the discrete nature of the variable (burst/no burst), the sample size needed to determine statistical power was too large. Hence, capsule burst was not used in this study as an indicator of contamination.Since the assay membrane is impermeable to Empty microcapsules and those containing 100 µL by volume of the X-Gal substrate  were used as negative and positive controls for catalytic activity. In past studies, empty microcapsules turned blue, if alginates were not sterilized, resulting in a false positive outcome as a result of yeast contamination . The conI) of the blue color was observed by the human eye referencing the scale presented in Biomolecular activity on the assay was monitored hourly or bi-hourly time intervals, before the optimum times for observation and data collection were executed. During optimization and confirmation experiments, the intensity (GI) values of a single well feature-extracted and imported into Excel. The intensity values were normalized (NI) according to the background intensity measured from an empty microcapsule (GI0) according to Equation (1): After 8 h, which was determined to be the maximum detection time to allow the color resolution at the upper end of the blue spectrum to be acceptable, a picture of the assay was taken using an iPhone camera. The image was uploaded to ImageJ version 1.51 , and grayscale intensities were determined. Shown in 16 Taguchi orthogonal array experiment was conducted with a “larger-the-better” optimization goal [BreastMilk Volume), lysis incubation time (Lysing Time), volume of X-gal substrate encapsulated in each capsule , concentration of E. coli cells (Contamination Level), and assay temperature (Temperature). Shown in In order to determine which variables had the greatest impact on the assay performance, an Lion goal . The expThe optimized level of parameters was determined by the difference (D), rank (R), signal-to-noise ratios (S/N), and gain (G) using the larger-the-better-optimization, which were given as following. D, was given by:X = S/N or X = I and I is the categorical intensity ranking based on Difference, R, was given by: i, j, and l correspond to the level, factor, run#, respectively, and L is the total number of levels.Rank, lth experiment (lS/N) was defined by n, the total number of replicates per experiment, and the observed color intensity for the mth trial of the lth experiment mjY,. Since runs were not replicated (m = n = 1), the equation for the average S/N was simplified as: The theoretical signal-to-noise ratio (dB) for the jw) for a given difference expressed in terms of signal-to-noise ratio, which was written as:The gain was defined as the sum of weighted ranks (D expressed in terms of intensity (I). If a solution to the optimization problem resided in the experimental space, the optimal level of variables was a compromise between the highest gain and the acceptable response The material cost for the individual assay was calculated by adding the amount of alginate, X-Gal, and lysing solution components.Statistical analysis was conducted using MATLAB v2019a . A one-sided student t-test for independent means at a significance level of 5% (α = 0.05) was conducted on feature-extracted color intensity measurements.Shown in I) and signal-to-noise ratio ranks, BreastMilk Volume and Contamination Level (CFU/mL) were the top two factors, with Temperature, X-Gal Volume, and Lysing Time ranking third, fourth, and fifth, respectively. Optimization results are summarized in E. coli was reported to be at either 37 [Lysing Time, the more the enzyme extracted across combinations of Contamination Level. According to For the volume of breast milk, 150 µL of samples enabled the highest level of expression, as the S/N increased with induction volume as illustrated in ither 37  or 50 °Cither 37 . Based oThe theoretical optimum levels of operation are presented in I) were re-examined for additional validation of theoretical analysis. Multiple optimal solutions as a combination of levels and variables were obtained as shown by the color change spectrum displayed in I = 8), which was chosen over run L (I = 8) for confirmation runs because of the higher rank of induction volume in the signal-to-noise ratio and pareto of effects. Furthermore, run N (I = 8) was not considered, because at 45 °C higher incubation times may affect the heat stability of the enzyme. The conditions of run G (I = 7.5) were not selected because of the lower gain. Hence, run M was chosen as the confirmation run.Before proceeding to the confirmation runs results of individual runs in terms of categorical intensity (I = 8), confirming the recommended conditions. The lack of resolution of the categorical scale at the upper limit did not enable the determination of a robust standard deviation.Run M was triplicated with an average intensity score of 8 . Statistical comparisons of normalized intensity values shown in 2 CFU/mL, positioning the LOD at 10 CFU/mL. However, there was no statistical significance between 102 and 103 CFU/mL (p = 0.2078), resulting in the poor linearity of the assay when the range was extended to 103 CFU/mL (R2 = 0.8581). Combining the analyses from both intensity scales, a binary LOD of 102 CFU/mL and above was determined. As reflected by the norm of the calculated The cost range per assay at the optimal X-Gal concentration was calculated to be between $0.32 and $0.81. The complete assay included the gel capsule prototype ($0.0003) and X-Gal ($0.0047), in conjunction with the B-PER lysing protocol ($0.32–$0.80). A unique key component of the assay was the entrapment of the X-Gal substrate in hydrogel to conserve the normal (unreacted/pre-diffused) and hydrolyzed (dimerized product diffused back into the microcapsule) conformations of X-Gal. In this case, alginate, a low-cost and well-researched biomaterial, was chosen as the entrapment gel. The maximal color change was observed after 8 h determined by multifactorial optimization. The composition of human milk is a dynamic variable ranging from 1 to 8 g/dL ,35. Sour3 CFU/mL. While using a single batch of bacteria and a unique sample of breast milk for LOD determination, the lack of linearity at the lower LOD can be attributed to the intensity normalization from the grayscale in the absence of a lookup table (LUT). The above root cause contributed to the variation in grayscale intensity at lower pathogen concentrations. At higher bacterial concentrations, these sources were masked by the contrast in the grayscale generated by the stronger intensity of blue color as a result of X-Gal dimerization. Combining future spectrophotometric absorbance measurements obtained from the assay supernatant and grayscale intensities will enable the correlation of pixel intensity to absorbance and hence the generation of a robust LUT. Furthermore, results will be collected at additional bacterial concentration increments in the range of 0–10Another hypothesis could be the nonuniform radial diffusion of the dimerized X-Gal into the capsule, leading to an uneven distribution of intensity between the capsule and the solution. Although this hypothesis can be refuted at higher concentrations as illustrated in Coupling spectrophotometric measurements to image analysis will shed light into whether a change in immobilization morphology for X-Gal is necessary.E. coli and/or other pathogens. In terms of cost, the assay has the potential to become a low-cost point-of-use assay, with costs ranging from $0.32 to $0.81, depending on the concentration of reagents used for the B-Per protein extraction method. In a parallel study for the confirmation runs of the L16 design, the use of the Miller protocol [® Bacteria Test Kit, which detects E. coli, Pseudomonas, and many other forms of bacteria in 15 min at less than $3 per test [Based on a literature review, this proposed hydrogel-based assay has a number of competitive advantages over technology currently in use for the detection of protocol  brought protocol . While tper test . 2 CFU/mL and above is approximately two orders of magnitude higher than the reported 0.093 CFU/mL for electrochemical immunoassays [E. coli contamination are significantly high for human breast milk banks, ranging from $35 to $81 to process approximately 100–200 ounces plus labor cost [As far as pathogen detection in biofluids across paper-based, microfluidic and electrochemical bioassays ,40,41,42noassays . Furthernoassays ,35,36,37noassays . Howevernoassays  to 24 honoassays . The capbor cost . In esseE. coli, Staphylococcus aureus, and group B 3-haemolytic streptococci are potentially pathogenic organisms that are present in high counts in pooled human milk [4 CFU/mL is implemented as a cutoff number for E. coli and S. aureus [It has been reported that man milk . Practicman milk ,46. Howeman milk , and in . aureus . In part. aureus . 2 CFU/mL and above, this proposed hydrogel-based assay is suited to meet requirements for screening breast milk or milk for pathogenic organisms with a percentage of false positives to be determined.Considering the reported binary LOD cutoff of 105 CFU/mL and lower), rapid detection (two–eight hours), low cost, environmental friendliness associated with the research objectives were achieved. Future efforts will encompass the following aspects: (1) examination of the effect of varying lactose concentration on induction by using various batches of breast milk; (2) use of other pathogenic bacterial strains commonly found as contaminants in breast milk; (3) optimization of lysing/extraction steps for reducing assay development time and costs, as well as increasing sensitivity; (4) development of a mobile app that can interpret and store detection results; (5) a twofold analysis of quantitative results from categorical to continuous scale with regards to image processing and automation with the aid of artificial intelligence; and (6) changes in assay morphology [By simplifying the colorimetric assay design from a silicone-based paper platform to a bioactive alginate microencapsulation platform, the specific aims of sensitivity (10rphology  guided b


An increasing number of protein vaccines have been researched for cancer, inflammation, and allergy therapies. Most of the protein therapeutics are administered through injection because orally-administered proteins are metabolized by the digestive system. Although transdermal administration has received increasing attention, the natural barrier formed by the skin is an obstacle. Monoolein is a common skin penetration enhancer that facilitates topical and transdermal drug delivery. Conventionally, it has been used in an aqueous vehicle, often with polyhydric alcohols. In the current study, monoolein was dissolved in an oil vehicle, isopropyl myristate, to facilitate the skin permeation of powder proteins. The skin permeabilities of the proteins were examined in-vivo and ex-vivo. Monoolein concentration-dependently enhanced the skin permeation of proteins. The protein permeability correlated with the zeta potential of the macromolecules. Dehydration of the stratum corneum (SC), lipid extraction from the SC, and disordering of ceramides caused by monoolein were demonstrated through Fourier transform infrared spectroscopic analysis and small-angle X-ray scattering analysis. An antigen model protein, ovalbumin from egg white, was delivered to immune cells in living mice, and induced antigen-specific IgG antibodies. The patch system showed the potential for transdermal vaccine delivery. Proteins as therapeutics include hormones, antibodies, and antigens that are necessary for the treatment of genetic disorders, autoimmunity/inflammation, cancers, and allergic diseases ,2. Protew/w) [Monoolein (MO) is a glyceryl 1-monoester of oleic acid , and is w/w) . The revw/w) , with thw/w) ,18. The Recently, the bioactive molecules were administered as powders dispersed in the delivery vehicle, to prevent the drug denaturation and to achieve the efficient delivery. The crystal reservoir technology employed in the tulobuterol patch  enables sustained drug release at a constant level, using a mixture of dissolved tulobuterol and tulobuterol crystals . In otheIn the current study, we have developed a needle-free protein delivery system composed of protein dispersion in an oil vehicle to facilitate the skin permeation of proteins from powder in an occlusive patch. Factors that could affect the skin permeation efficiencies of proteins were examined, such as the MO concentration, the zeta potential, and the hydrodynamic diameter of proteins. The IPM dispersion of an antigen model protein, ovalbumin from chicken egg white (OVA), was applied to shaved mouse back skin. As a result of the skin permeation enhancing effect of MO, the oil-based patch system elicited a high level of serum antigen-specific antibodies.OVA (43 kDa), FITC-conjugated cholera toxin subunit B pentamer (11.6 kDa each), lysozyme from chicken egg white (14 kDa), and FITC-conjugated dextrans  were purchased from Merck . Horseradish peroxidase (44 kDa) and bovine serum albumin (66 kDa) were obtained from FUJIFILM Wako Chemicals . Green fluorescent protein (27 kDa) was a generous gift from Prof. K. Minamihata, Kyushu University. N-terminal 5-FAM-labeled peptide  was custom synthesized at GenScript . IPM and trypsin (0.5 g/L)-ethylenediaminetetraacetic acid (0.53 mmol/L) solution were purchased from Nacalai tesque . MGOL-70 was kindly supplied by Nikko Chemicals . Frozen YMP skin was obtained from Charles River Laboratories  and stored at −80 °C until use.Female BALB/c mice (six week old) were purchased from Kyudo , and maintained under pathogen-free conditions. All animal experiments were approved by the local Ethics Committee for Animal Experiments .Proteins and FITC-dextrans  were dissolved in PBS. Hydrodynamic diameters and zeta potentials were measured using a Zetasizer NanoZS . w/w).OVA was dissolved in Milli-Q water (10 mg/mL) and a 100 μL aliquot was added in a 1.5-mL tube, and then freeze-dried overnight. Liquid MGOL-70  was added to the freeze-dried OVA powder (1 mg), and they were mixed by vortexing for 10 s. Then, dehydrated IPM  was added to the OVA−MGOL-70 dispersion and mixed well using a vortex mixer for 60 s to obtain an OVA dispersion in MO and IPM  , supported by a waterproof-breathable backing with an acrylic adhesive (2.5 × 2.5 cm2)  to prepare a patch. The resulting OVA dispersion (50 μL) was penetrated into a polyurethane foam sheet  according to the manufacturer’s instructions and was loaded in the patch in the same manner. 2). The YMP skin stored at −80 °C was thawed and the subcutaneous fats and tissues were carefully removed using a scalpel. Then, the skin was cut into 1.5 × 1.5 cm2 pieces and placed onto the receptor compartment. The receptor compartment was filled with PBS and maintained at 32 °C. FITC-labeling of proteins was performed as previously reported [The ex-vivo skin permeability of the OVA was examined using Franz diffusion cells  with and without MGOL-70 (25 mg/mL) were prepared and stored for 24 h at room temperature. An aliquot (200 μL) was carefully added to PBS (1 mL) and stirred with a magnetic stirrer for 24 h to let the OVA molecule dissolve. The protein concentration in PBS was determined at 280 nm and adjusted to be 0.1 mg/mL with PBS. The secondary structures of OVA in the PBS (0.1 mg/mL) were analyzed using a circular dichroism spectrometer J-725G  at 25 °C with a 1 mm cell. The percentage of the α-helical structure was calculated using the equation reported by Cheng and Yang [2 pieces. The skin pieces were placed in a plastic dish and immersed in a trypsin (0.5 g/L)-EDTA (0.53 mmol/L) solution. The skin was incubated overnight at 30 °C, and then the SC was peeled off. The SC was rinsed with Milli-Q water, air-dried, and placed on a glass plate, SC side up. A patch was applied to the SC surface for 24 h at 32 °C. After removal of the patch, the SC sample was rinsed with Milli-Q water, 80% ethanol, and then Milli-Q water (5 mL each) again, and then immediately wrapped in a plastic film and stored on ice until analysis. The SC was isolated from YMP skin as reported previously ,26. BrieSmall-angle X-ray scattering (SAXS) patterns were recorded using a NANOSTAR system , with CuKα radiation at 50 kV and 100 mA. The SC sample was attached to the film holder with Kapton tapes. The film holder was equipped with a water heating system adjusted to 32 °C. SAXS patterns were scanned for 2 h under atmospheric pressure. −1, with 64 scans at room temperature. An air background spectrum was withdrawn from each spectrum. Fourier transform infrared (FT-IR) spectra were measured using a Spectrum Two equipped with a Universal Attenuated Total Reflectance accessory . The FT IR spectra of SC were measured at a resolution of 2 cmMouse hair was removed using depilatory cream 24 h prior to OVA administration. The patch apparatus was applied onto the back skin (n = 3). After 24 h, the patches were removed and wiped with isopropanol, and OVA diffusion was observed using an IVIS Lumina II . Mice were sacrificed and the skin areas where the samples were attached were collected. The skin was cut into halves and confocal images were obtained using a Confocal Laser Scanning Microscope LSM700 with a 20× objective lens . Half of the skin was embedded in Tissue-tek O.C.T. compound, frozen in liquid nitrogen, and then sectioned at a 16-μm thickness using a cryostat CM1860UV . The skin sections were observed using a fluorescence microscope BZ-9000 with a 20× objective lens .Freeze-dried OVA (1 mg) was dispersed in MGOL-70 (25 mg) and then IPM (0.5 mL) for the immunization of the mice. An aliquot  was loaded onto the patch apparatus, and then placed onto the mouse back skin for 24 h, once a week for 3 w (n = 6). A total of 2 w after the final administration, blood samples were collected for sera. PBS solutions of OVA  were similarly applied onto the mouse back skin as controls. A serum OVA-specific IgG titer was measured as previously reported .p < 0.05).Statistical analysis: OVA-specific IgG levels were analyzed for the distribution of the oil-based delivery system using Student’s t test  to function as a skin penetration enhancer of hydrophilic biomacromolecules, proteins and dextrans. The skin permeability of OVA was increased depending on the concentration of MO up to 6% w/w A. Less Ow/w. The skin permeation efficiency was strongly affected by the zeta potential of the protein, rather than by the hydrodynamic diameter of the protein  is composed of 30.8% α-Helix, 31.3% β-Strand, and 16.4% Turn . In the −1 derived from water molecules through treatment with the oil-based patches  and 1398 cm−1 (δCH2) disappeared [−1 to 800 cm−1 in the MO and IPM spectra were observed in the spectrum of the SC treated with the oil-based patches, indicating penetration of these substances into the SC. The peaks from 1440 cm−1 to 1470 cm−1 became sharp, indicating the disordering of ceramides from orthorhombic perpendicular packing  spectrum is an indicator of the secondary structure of a protein . In the w/w. In previous studies, the periodic X-ray diffraction pattern derived from the SC disappeared following dehydration [−1 to 1470 cm−1 derived from the symmetric CH2 bending indicated disordering of the ceramide orthorhombic perpendicular packing following oil-based patch application [2 stretching region from lipids (2850–2925 cm−1) indicates the alignment and the fluidity of the lipids [2 stretching occurred from 2852 cm−1 to 2856 cm−1 indicated an increase of the lipid fluidity in the SC caused by monoolein. No shift was observed in the asymmetric CH2 stretching peak, however, sharpening of the peak at 2928 cm−1 indicated infiltration of IPM into the SC.Although MO did not denature the OVA, it was possible that it affected the lipids in the SC. Based on the small angle X-ray scattering patterns, MO affected the lamellar structure in the SC at 3% ydration , and thrydration . In the ydration ,40. The lication . The syme lipids . The shiAs mentioned above, MO is non-toxic. However, repeated overuse may cause skin irritation, as with oleic acid . In the The oil-based patch system elicited significantly higher levels of antigen-specific IgG antibodies than the aqueous-based patch system. The skin thickness differs between mice and humans, and the human skin thickness and structure are more similar to those of the YMP skin. Since OVA permeated through to the middle of the dermis in the YMP skin, and not to the receptor phase after 24 h, the efficiency of the oil-based patch on human skin needs to be investigated. However, APCs exist in the human epidermis, and some techniques, such as epicutaneous immunotherapy, successfully achieved modification of antibody responses by delivering the antigen proteins to the epidermis ,22. A trAn MO-assisted oil-based occlusive patch to deliver antigen powder to the skin was investigated. The protein permeability increased along with increasing MO concentrations in the IPM vehicle, indicating the skin permeation enhancement of MO. The proteins with higher negative zeta potentials more easily permeated into the skin. The effect of MO in our oil system was suggested to be limited to SC. Any serious adverse events were not observed during patch application on mouse back skin for 24 h. The oil-based system induced significantly higher levels of antigen-specific IgG than that of the aqueous system, indicating the potential of the oil-based patch system for a non-invasive transdermal vaccine delivery.